Shortly after the first strains of dengue virus were isolated in the laboratory, serial intracerebral passage of dengue type 1 or type 2 virus (DEN1 or DEN2) in mice was employed to attenuate these viruses for use as live attenuated vaccines in humans. Studies by Sabin revealed that at a low passage level these viruses had lost most of their virulence. In addition, a highly neurovirulent mutant of the non-pathogenic parental DEN4 strain H241 was selected by serial intracerebral passage in mice. The genetic basis for neurovirulence of the DEN4 mouse-adapted mutant was studied by comparing intratypic chimeric viruses that contained the three structural protein genes of the parental virus or its neurovirulent mutant on the background sequence of non-neurovirulent DEN4 strain 814669. The chimera that contained the three structural protein genes of mouse neurovirulent DEN4 H241 was highly neurovirulent in mice, whereas the chimera that contained the corresponding genes of its non- mouse adapted parent was not neurovirulent. Thus, some or all of the genetic loci for neurovirulence of the DEN4 mutant map within the structural protein genes. The parent and its mouse neurovirulent mutant were found to differ by only five amino acid differences in their structural proteins. Three of the mutations were located in the envelope (E) glycoprotein. Two of these amino acid changes were identified as important determinants of DEN4 mouse neurovirulence: (i) the single substitution of Ile for Thr434 which ablated one of the two conserved glycosylation sites in DEN4 E yielded a virus that was almost as neurovirulent as the mouse-adapted mutant; and (ii) the Leu for Phe680 substitution also yielded a neurovirulent virus but it was less neurovirulent than the glycosylation mutant. The parental DEN1 Hawaii strain and its mouse neurovirulent mutant were also studied in an attempt to map the DEN1 genetic loci responsible for mouse neurovirulence. Thirteen amino acid differences in the C-PreM-E structural protein region were identified. Among the 13 changes 7 are located in E, 3 in PreM and 3 in C.